Campylobacter spp. is a leading cause of acute gastroenteritis globally, with significant risks for invasive infections and post-infection sequelae like Guillain-Barré Syndrome (GBS). In India, campylobacteriosis remains understudied, with prevalence estimates varying widely. This study aimed to assess the prevalence of Campylobacteriosis and the clinical presentation in patients with acute gastroenteritis across North India. We compared the performance of culture, real-time PCR, and ELISA in detecting Campylobacter jejuni and Campylobacter coli, emphasising the need for more efficient diagnostic methods to support surveillance and risk assessment in India. This study aimed to determine the prevalence of Campylobacter in acute diarrhoea cases across five states in North India, using both culture- and culture-independent methods. A high prevalence (11.18%) was observed, surpassing previous regional reports. This study revealed that Campylobacter infections are not limited to young children but are common in adults (>40 years), with a notable second peak in this age group. Most cases were mild to moderate, with severe infections occurring in 19.3%. We compared three detection methods: culture, ELISA and real-time PCR, finding that real-time PCR had the highest sensitivity (87.0%) and specificity (92.0%), followed by ELISA (78.26% sensitivity and 91.1% specificity). PCR and ELISA identified an additional 8% of cases missed by culture, highlighting the limitations of culture methods. This study emphasises the need for molecular techniques to improve the detection and surveillance of Campylobacter, especially given its association with GBS, which has been linked to 20 to 30% of cases in a current outbreak in India. Enhanced diagnostic tools will strengthen India’s surveillance infrastructure and improve responses to Campylobacter-related gastroenteritis and GBS. This study provides insights into the prevalence and epidemiology of Campylobacter in community-acquired diarrhoea in North India. Our findings highlight the poor sensitivity of culture methods and emphasise the need to combine culture with either ELISA or real-time PCR for accurate clinical diagnosis. This large-scale study reveals that Campylobacter contributes to a higher proportion of diarrhoea cases than previously reported, with most infections being mild to moderate in children and adults.

Amplicon sequencing is a popular method for understanding the diversity of bacterial communities in mixed samples as exemplified by 16S rRNA metagenome sequencing. This approach has been extended into multiplex amplicon sequencing in which multiple targets are amplified in the same polymerase chain reaction (PCR). Multiple tools exist to process the sequencing data produced via the short-read Illumina platform, but there are fewer options for long-read Oxford Nanopore Technologies (ONT) sequencing, or for processing data from environmental surveillance or other sources with many different organisms. We have developed AmpliconTyper (v0.1.28, DOI: 10.5281/zenodo.14621928) for analysing multiplex amplicon sequencing data from environmental (e.g. wastewater) or similarly contaminated samples, generated using ONT devices. The software tool uses machine learning to classify sequencing reads into target and non-target organisms with very high specificity and sensitivity. The user can train models using public and/or user-generated data, which can subsequently be applied to analyse new data. The tool can also generate amplicon consensus sequences, as well as identify single nucleotide polymorphisms (SNPs) and report their genotype implications, such as association with lineages or antimicrobial resistance (AMR). The tool is freely available via Bioconda and GitHub (https://github.com/AntonS-bio/AmpliconTyper). AmpliconTyper allows robust identification of target organism reads in ONT sequenced environmental samples, and can identify user-specified lineage or AMR markers. ### Competing Interest Statement The authors have declared no competing interest.

The following protocol is for use on environmental samples that have been collected by Moore swab or grab sampling methods and processed up to the stage of DNA extraction. The protocol includes DNA extraction and qPCR analysis of the samples to detect three gene targets for identifying Salmonella Typhi, a human faecal indicator (HF183), and a commercial spike-in control. The qPCR uses fluorescence of Taqman probes for measuring the product. Care must be taken to ensure that the fluorescent dyes chosen for the probes are suitable for the machine being used or can be calibrated for use on the machine. If the dyes selected are different from those described in the protocol, make sure that their excitation/emission spectra do not overlap to avoid crossover between targets. Generation of standard curves is mentioned in this protocol but is described in more detail in a separate protocol in the Typhoid ES workspace.

The following protocol describes the resuspension, dilution, and qPCR of gBlocks gene fragments. gBlocks gene fragments are synthesised double stranded DNA oligos, which can be used for standardisation. In this protocol, the standard curves generated by the gBlocks in a triplex qPCR are used to determine a Ct cut-off value for the S. Typhi gene targets (ttr, tviB, staG, C6) and the HF183 Bacteroides rRNA gene.

Amplicon sequencing is a popular method for understanding the diversity of bacterial communities in samples containing multiple organisms as exemplified by 16S rRNA sequencing. Another application of amplicon sequencing includes multiplexing both primer sets and samples, allowing sequencing of multiple targets in multiple samples in the same sequencing run. Multiple tools exist to process the amplicon sequencing data produced via the short-read Illumina platform, but there are fewer options for long-read Oxford Nanopore Technologies (ONT) sequencing, or for processing data from environmental surveillance or other sources with many different organisms. We have developed AmpliconTyper (v0.1.28, DOI: 10.5281/zenodo.15045111) for analysing multiplex amplicon sequencing data from environmental (e.g. wastewater) or similarly complex samples, generated using ONT devices. The tool uses machine learning to classify sequencing reads into target and non-target organisms with very high specificity and sensitivity. The user can train models using public and/or user-generated data, which can subsequently be applied to analyse new data. The tool can also generate amplicon consensus sequences, as well as identify SNPs and report their genotype implications, such as association with lineages or antimicrobial resistance (AMR). The tool is freely available via Bioconda and GitHub (https://github.com/AntonS-bio/AmpliconTyper). AmpliconTyper allows robust identification of target organism reads in ONT-sequenced environmental samples and can identify user-specified lineage or AMR markers.

Background Amplicon sequencing is a popular method for understanding the diversity of bacterial communities in environmental or similar samples as exemplified by 16S rRNA sequencing. This approach has been extended into multiplex amplicon sequencing in which multiple targets are amplified in the same PCR reaction such as virus sequencing using tiled amplicons. Multiple tools exist to design PCR primers, and some support design of multiplex panels. However, despite increasing interest in the use of environmental or wastewater sampling for detecting and typing specific antimicrobial resistant (AMR) bacteria such as typhoid or cholera agents, we were unable to find a tool for designing a multiplex PCR panel for environmental samples that not only focused on detection of a specific organism, but also on amplifying lineage-specific or AMR-associated alleles of the target organism while minimising amplification of off-target genomes present in the sample. We found that existing tools either depend on the target organism being very distinct from the rest of the organisms in the sample, or focus on detection rather than genotyping of the organism of interest. We have developed EnviroAmpDesigner (v0.1.3, DOI: 10.5281/zenodo.14967337) to fill this gap, which we used to design a multiplex amplicon panel for detection, genotyping and AMR profiling of the typhoidal pathogens Salmonella enterica serovars Typhi and Paratyphi A. EnviroAmpDesigner can design amplicons for both short and long-read sequencing. The software first identifies single nucleotide polymorsphisms (SNPs) that distinguish individual genotypes of the target pathogen (in our case, S . Typhi). It then identifies SNPs that distinguish the target organism from others. Finally, the software designs primers that simultaneously target genotyping SNPs and have at least one primer with a 3’ end at each SNP that distinguishes the target organism. The purpose of the latter condition is to increase the specificity of the primers, to minimise amplification of homologous sequences in non-target organisms. While our use case focuses on S . Typhi and Paratyphi A, the tool is organism agnostic and should work for any haploid organism. The tool is freely available via Bioconda and GitHub (). Impact statement We have developed EnviroAmpDesigner (v 0.1.3) software to design a multiplexed panel of long-read amplicons for detection and lineage typing of bacteria in environmental samples. We apply this tool to Salmonella Typhi and Paratyphi A, the bacterial agents of typhoid fever, but the tool is organism agnostic. The tool is computationally efficient and can be used on mid-range laptop. It has potential to support more widespread deployment of sequencing-based surveillance for bacterial pathogens in environmental and wastewater samples. Data Summary For the purposes of designing primers for S . Typhi and Paratyphi A we used 13,134 samples from the Global Typhoid Genomics Consortium data (target organisms) ([1][1]) and 1,810 publicly available Enterobacterales genomes from NCBI’s RefSeq database (representing off-target related organisms) ([2][2]). To validate the primers, we applied an amplicon sequencing protocol ([3][3]) to DNA extracted from two S . Typhi isolates (NCBI accessions SRR5949979 and SRR7165748) provided by Satheesh Nair (UKHSA) ([4][4]), and a pooled DNA sample of American Type Culture Collection (ATCC) isolates S . Paratyphi A (ATCC 9150D), S . Paratyphi B (ATCC-BAA-1250D), S . Paratyphi C (ATCC-BAA-1715D), Aeromonas hydrophila (ATCC-7965D), Klebsiella pneumoniae (ATCC-BAA-1706D), and Citrobacter freundii (ATCC-8090D) chosen for their close relationship to S . Typhi. The resulting amplicon sequence data was deposited in European Nucleotide Archive (project accession: PRJEB81565). ### Competing Interest Statement The authors have declared no competing interest. * ATCC : American type culture collection AMR : antimicrobial resistance BAM : Binary Alignment Map NCBI : National Center for Biotechnology Information ONT : Oxford Nanopore Technologies PCR : Polymerase chain reaction RT-qPCR : quantitative reverse transcription polymerase chain reaction SNP : single nucleotide polymorphism UKHSA : United Kingdom Health Security Agency VCF : Variant Call File WGS : whole genome sequencing Bill & Melinda Gates Foundation, https://ror.org/0456r8d26, INV047158 [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4

Klebsiella pneumoniae (Kp) associated with hospital acquired infections are extensively-drug resistant (XDR), making treatment problematic. Understanding the genetic epidemiology of XDR -Kp can determine their potential to be hypervirulent (hv) through the presence of siderophores. We characterized genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated urinary tract infection in an Indian healthcare facility. 18 organisms comprised STs: ST14 (9/18), ST147 (5/18), ST231 (2/18), ST2096 (1/18), and ST25 (1/18). Many patients in one ward were infected with the same ST, indicating a common infection source. Some patients had recurrent infections with multiple STs that were circulating in that ward, providing evidence for hospital transmission. Beta lactamase genes (blaCTX-M-1, blaSHV, and blaampH) were present in all isolates. blaNDM-1 was present in isolates 15/18, blaOXA-1 was present in isolates 16/18, blaTEM-1D was present in 13/18 isolates, and blaOXA-48 was present in isolates 14,19 and 30. Disruption of mgrB with various IS elements was responsible for colistin resistance in 6 isolates. The most common K type among these isolates was K2 (10/18). One XDR convergent hv-Kp ST2096 was associated with prolonged hospitalisation (iuc+ybt+blaOXA-1+blaOXA-48).Convergent XDR-hv-Kp detected has outbreak potential, warranting effective antimicrobial stewardship and infection control.

In the current study, we report whole genome sequencing (WGS) data on EAEC strains from India to identify lineages and different sequence types (STs) in our geographical regions across North India. We performed WGS comparative genomics characterization to examine the diversity of 122 EAEC strains collected from a large geographic area from clinical (Human sources) and non-clinical sources (animal and environmental sources). MPCR for 21 virulence genes was performed. A triplex PCR detected phylogenetic groups A, B1, B2, and D was done. All strains were genome-sequenced, and bioinformatics analysis was performed. EAEC isolates belonged to 29 sequence types, further clustered into 11 clonal complexes, among which CC38 was the largest, containing 38 isolates mainly belonging to two ST types (ST38 and ST315). CC10 was the most diverse group, comprising 8 STs (ST43, ST2706, ST1286, ST 10, ST167, ST34, ST227, and ST4305). The most frequently detected virulence gene among the 96 clinical EAEC isolates was astA (87.5%), followed by ORF3 (62.5%), and aap (54.1 %). ST131, known for multidrug resistance and causing various diseases, was detected in acute diarrhea cases and animal sources, underscoring its clinical significance. These findings indicate the high diversity of EAEC and different sources of unique ST types of EAEC. The study reinforces the One Health approach, highlighting the interconnection between human health, animal reservoirs, and environmental sources in EAEC transmission dynamics. The identification of EAEC strains in animals suggests the zoonotic potential of these pathogens, necessitating comprehensive surveillance and control measures. The study emphasizes the need for continued surveillance and a One Health approach to address the complex transmission dynamics of EAEC.

Introduction: Non-typhoidal Salmonella (NTS) serovars are the leading global cause of gastroenteritis and have established reservoirs in food animals. Gap statement: Due to a lack of surveillance, there is limited information on the distribution of NTS serovars in India. Aim: Here, we investigated the epidemiology, sequence types, serovar distribution, phylogenetic relatedness, and antimicrobial resistance patterns of NTS in humans and animals across a large geographic area in Northern India. Methodology: We collected stool samples from patients with diarrhea who presented to 14 laboratories in Chandigarh and from five states in India (Punjab, Haryana, Uttarakhand, Himachal Pradesh, and Rajasthan). We sequenced the genomes and analyzed 117 NTS organisms isolated from humans and animals. Minimum inhibitory concentrations (MICs) were estimated using a Vitek2 system. Results: The prevalence of NTS in participants presenting to our study with diarrhea was 1.28 %, affecting all age groups. All NTS caused moderate to severe diarrhea. We found a high diversity of serovars with considerable serovar and sequence types (STs) overlap and phylogenetic closeness between isolates from human infections and food animals. We report serovars such as S. Agona, S. Bareilly, S. Kentucky, S. Saintpaul, and S. Virchow, causing human infections from north India for the first time. Among the different food-producing animals, pigs appeared to be a key source of human infections. Twenty-eight percent (28 %) of the NTS isolates were multi-drug resistant (MDR), and human isolates showed a higher proportion of resistance. A higher level of contamination of meat samples in our study (8.4 %) potentially suggests a close association of NTS serovars with the food chain and high transmission risk in north India. Conclusions: This study provides information on AMR genes and plasmid replicons associated with different serovars and highlights the role of food animals in AMR dissemination in our region.

This protocol is for direct detection of Salmonella Paratyphi A in wastewater. The wastewater extract can be pre-screened by qPCR for possible presence of Salmonella using triplex qPCR as described in https://doi.org/10.1093/infdis/jiad427

Background: The burden of foodborne diseases and antimicrobial resistance carried by key foodborne pathogens in India is unknown due to a lack of an integrated surveillance system at the human-animal interface. Methods: We present data from the WHO-AGISAR (Advisory Group on Integrated Surveillance of Antimicrobial Resistance), India project. Concurrent human and animal sampling was done across a large area across north India. Community-acquired diarrhea cases (n = 1968) of all age groups were included. Cross-sectional sampling of stool/ intestinal contents (n = 487) and meat samples (n = 419) from food-producing animals was done at farms, retail shops, and slaughterhouses. Pathogens were cultured and identified, and antimicrobial susceptibility was performed. Results: Over 80% of diarrhoeal samples were obtained from moderate to severe diarrhea patients, which yielded EAEC (5%), ETEC (4.84%), EPEC (4.32%), and Campylobacter spp. (2%). A high carriage of EPEC (32.11%) and Campylobacter spp. (24.72%) was noted in food animals, but the prevalence of ETEC (2%) and EAEC (1%) was low. Atypical EPEC (aEPEC, 84.52%, p <= 0.0001) were predominant and caused milder diarrhea. All EPEC from animal/poultry were aEPEC. Overall, a very high level of resistance was observed, and the MDR rate ranged from 29.2% in Campylobacter spp., 53.6% in EPEC, and 59.8% in ETEC. Resistance to piperacillin-tazobactam, cefepime, ceftriaxone, and co-trimoxazole was significantly higher in human strains. In contrast, resistance to ciprofloxacin, aminoglycosides, and tetracycline was higher in animal strains, reflecting the corresponding usage in human and animal sectors. ESBL production was commoner in animal isolates than in humans, indicating high use of third-generation cephalosporins in the animal sector. C. hyointestinalis is an emerging zoonotic pathogen, first time reported from India. Conclusion: In one of the most extensive studies from India, a high burden of key foodborne pathogens with MDR and ESBL phenotypes was found in livestock, poultry, and retail meat.

The following protocol is for amplifying and sequencing amplicons targeting Salmonella Typhi. It is primarily for use with samples that are already suspected to be positive for S. Typhi and has been designed for use with DNA extracted from environmental surveillance samples. The resulting sequences can be analysed and used for genotyping (given that the genotype is targeted by the primer panel) and determining antimicrobial resistance. The genotypes targeted in the panel in this protocol are listed but primers can be designed for other S. typhi genotypes of interest. The library preparation steps of this protocol are adapted from the Oxford nanopore protocol "Ligation Sequencing Amplicons - Native Barcoding Kit 24 V14 (SQK-NBD114.24)" which is available on the Nanopore community.

The following protocol is for amplifying and sequencing amplicons targeting Salmonella Typhi. It is primarily for use with samples that are already suspected to be positive for S. Typhi and has been designed for use with DNA extracted from environmental surveillance samples. The resulting sequences can be analysed and used for genotyping (given that the genotype is targeted by the primer panel) and determining antimicrobial resistance. The genotypes targeted in the panel in this protocol are listed but primers can be designed for other S. typhi genotypes of interest. The library preparation steps of this protocol are adapted from the Oxford nanopore protocol "Ligation Sequencing Amplicons - Native Barcoding Kit 24 V14 (SQK-NBD114.24)" which is available on the Nanopore community.

Salmonella enterica Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the causative agents of enteric fever, a systemic human disease with a burden of 300 000 cases per year in India. The majority of enteric fever cases are associated with S. Typhi, resulting in a paucity of data regarding S. Paratyphi A, specifically with respect to genomic surveillance and antimicrobial resistance (AMR). Here, we exploited whole-genome sequencing (WGS) to identify S. Paratyphi A genotypes and AMR determinants associated with an outbreak of S. Paratyphi A in Vadodara, India, from December 2018 to December 2019. In total 117 S. Paratyphi A were isolated and genome sequenced, most were genotype 2.4.2 (72.6 % of all cases), which is the globally dominant genotype. The remainder were genotype 2.3 (25.6 %), while only two isolates belonged to genotype 2.4.1. A single base-pair mutation in gyrA, associated with reduced susceptibility to fluoroquinolones, was present in all of the outbreak isolates; with 74.35 % of isolates having a S83F substitution and the remainder having an S83Y substitution. Our surveillance study suggests that S. Paratyphi A is an emergent pathogen in South Asia, which may become increasingly relevant with the introduction of Vi conjugate vaccines.

Introduction: Diarrheagenic Escherichia coli (DEC) are responsible for about 30–40% of acute diarrhoea episodes in developing countries. Up to 10% of isolates of E. coli from stool specimens have been reported to be non-lactose fermenting (NLF E. coli). There is scarcity of literature available regarding the pathogenicity of NLF E. coli causing infectious diarrhoea. Aim: We aimed to elucidate the importance of NLF E. coli in causing diarrhoea in both adults as well as children by detecting various DEC pathotypes among NLF E. coli in stool samples from gastroenteritis cases. Material and Methods: A total of 376 non-lactose fermenting (NLF) E. coli isolates isolated from 3110 stool samples of diarrhoea/ gastroenteritis patients were included in the study. Upto 3 non-lactose fermenting colonies which did not confirm as V.cholerae, Aeromonas spp., Salmonella spp., Shigella spp., but were identified as E.coli on MALDI-TOF were selected from each MacConkey agar plate and streaked onto fresh, sterilized nutrient agar and biochemical reactions were put up. Multiplex PCR was put up for EAEC, EPEC, ETEC, EHEC pathotypes and PCR for ipaH gene was put up for EIEC. Antibiotic sensitivity testing was performed with the disc diffusion method. Results: Using multiplex PCR and ipaH PCR, a total of 63 pathotypes of DEC were obtained, EAEC being the most predominant (n= 31) followed by EIEC (n=22), EPEC (n=8) and ETEC (n=2). To further differentiate EIEC from Shigella, additional biochemicals were performed which include acetate utilization, mucate and salicin fermentation, and esculin hydrolysis. Antimicrobial susceptibility testing (AST) showed that maximum resistance was seen against ciprofloxacin (82.5%) followed by ampicillin (77.8%) and cotrimoxazole (68.2%) and minimum resistance was seen against ertapenem (4.8%). Conclusion: In our study two pathotypes (EAEC, EIEC) were predominant among NLF E. coli and these were important etiological agents not only in children but also in adults. Our study also sheds light on epidemiology of EIEC which is one of the most neglected DEC pathotype as hardly any microbiological laboratory process NLF E. coli for EIEC.

Background: The diminishing antimicrobial options for the treatment of XDR and PDR Acinetobacter baumannii is an increasing concern. In this study, we assessed the in vitro synergy of the fosfomycin (FOS) with meropenem (MEM), amikacin (AK), tigecycline (TGC), and colistin (CL) in whole genome sequenced isolates. Methods: Non-replicate whole genome sequenced (illumina next-generation sequencing platform, Clevergene, India), A. baumanii (7 XDR, 1PDR) were subjected to in vitro synergy testing by checkerboard (CB) and time kill assay (TKA) after MIC determination, with glucose-6-phosphate being incorporated in all runs. FOS was used as a cornerstone drug in four combinations and colistin in one. ResFinder, MLST, PlasmidFinder, and CSIPhylogeny tools were used. Results: Mortality occurred in three patients. Diverse MLST were observed, ST-1962 (3 isolates) and one each of ST2062, ST2063, ST1816, ST1806, ST234. FOS MICs ranged from 32 to 128 mg/L, MEM MIC: 16–64 mg/L, TGC MIC: ≤2–≤4 mg/L and AK MIC: >512 mg/L. CL: MIC range, 0.25–≤2 mg/L, PDR MIC > 16 mg/L. Synergy results by CB: FOS-MEM: synergy in ⅞ (90%) isolates. Synergy lowered MEM MICs to susceptibility breakpoints in 6/8 cases. CL-MEM: Excellent synergy (3/3) isolates. FOS-AK: Indifference in ⅞, antagonism ⅛ (AK-susceptible isolate). FOS-TGC: Partial synergy (PS) in 8/8 (TGC MIC dropped to ≤0.25 mg/L in 3/8). In the PDR isolate, synergy was seen in FOS-MEM, CL-MEM, PS in FOS-CL, FOS-TGC, indifference in FOS-AK. TKA: Excellent synergy was observed with FOS-MEM from 4 h, while FOS-AK and FOS-TGC demonstrated synergy at 24 h. Synergy was achieved despite presence of widespread resistance markers against aminoglycosides (AacAad, AadA, AadB, Aph3″Ia, ArmA, Arr, StrA, StrB), beta-lactams (ADC, BlaA1, BlaA2, Zn-dependent_hydrolase, OXA-23, OXA-51, PER-1,TEM-1D, CARB-5, Mbl), sulphonamides (SulII, SulI), phenicols (CatBx, CmlA), macrolides (MphE, MsrE) and tetracycline (TetB) were widespread. Carbapenemase, CARB-5 was present in one isolate. Beta-lactamase genes OXA-23, OXA-51, BlaA2, Zn-dependent_hydrolase, ADC, Mbl and macrolide resistance genes MphE, MsrE were present in all 8 isolates. Conclusions: FOS-MEM and CL-MEM are promising combinations against A. baumannii. Synergy of FOS-MEM in intrinsically resistant A. baumannii shows that this antibiotic combination might be useful in treating such XDR and PDR pathogens.

PURPOSE:Emergence and spread of resistance among Vibrio cholerae have become a global public health problem. In India, no consolidated data is available on antimicrobial susceptibility patterns and antibiotic resistance genes.METHODS:A total of 110 representative isolates obtained over a period of 14 years were included. Antimicrobial susceptibility was tested by disc diffusion and micro broth dilution. Presence of 13 antimicrobial resistance genes was ascertained by using PCR.RESULTS:Antimicrobial resistance fluctuated for most of the antibiotics. Resistance to cotrimoxazole in our study was 92.72% and the SXT element was present in all isolates. Resistance to nalidixic acid, tetracycline, and cefotaxime was found to be 98.18%, 7.27%, and 10.9% respectively. Resistance to ampicillin saw a fluctuating trend with a recent fall. Resistance to ciprofloxacin and azithromycin was 12.72% and 29% by MIC. blaTEM was the most common ESBL gene (94.5%). Other were blaCMY (26.36%) and blaNDM (2.7%). We report blaCTX-M-15 and blaOXA-48 and ermB for the first time in the world. Newer antimicrobials like prulifloxacin and rifaximin were tested for the first time from India.CONCLUSIONS:Our study has shown very high levels of resistance to older antibiotics and the emergence of resistance to some of the newer classes of antibiotics. There is an urgent need for increased surveillance studies, rational use of the antimicrobials and preventive measures to control the disease.

OBJECTIVE:To study the significance of enteroaggregative Escherichia coli (EAEC) as a pathogen causing acute diarrhea and a commensal in healthy nourished and malnourished children younger than five years of age in the Chandigarh region and to address possible traits of EAEC virulence genes, biofilm formation, phylogroups, and antibiotic resistance that would be correlated with diarrhea or carriage. STUDY DESIGN:Stool samples were obtained from children with acute diarrhea (n = 548), as well as nourished (n = 550), and malnourished controls without diarrhea (n = 110). E coli isolates were confirmed as EAEC by pCVD432 polymerase chain reaction. Multiplex polymerase chain reactions were used to identify 22 virulence-related genes and phylogeny. Antibiotic susceptibility, adherence, and biofilm-forming potential also were studied. RESULTS:Overall, 16.6% of children were malnourished. EAEC detection was greater among children with acute diarrhea (16%) than nourished (6%) and malnourished nondiarrheal controls (2.7%). We found an association of EAEC infections with age <2 years (P = .0001) in the diarrheal group. Adhesive variants adhesion fimbriae IV and adhesion fimbriae II were significantly associated with diarrhea. The aggR and aar genes showed a positive and negative association with the severity of disease (P = .0004 and P = .0003). A high degree of multidrug resistance was found (73.8%) in the diarrheal group. Most EAEC strains from the diarrheal group belonged to B2 and D phylogroups, whereas strains from non-diarrheal groups, which belonged to phylogroup B1. CONCLUSIONS:EAEC is a significant contributor to childhood diarrhea, its presence as a commensal, and the significance of the association of various virulence factors among the EAEC isolated from diarrheal and non-diarrheal stools. These data reinforce the importance of aggR and aar as positive and negative regulators and the contribution of AAF/II and AAF/IV fimbria for the pathobiology of EAEC.

Background. The emergence and spread of antimicrobial resistance (AMR) pose a major threat to the effective treatment and control of typhoid fever. The ongoing outbreak of extensively drug-resistant Salmonella Typhi (S. Typhi) in Pakistan has left azithromycin as the only remaining broadly efficacious oral antimicrobial for typhoid in South Asia. Ominously, azithromycin-resistant S. Typhi organisms have been subsequently reported in Bangladesh, Pakistan, and Nepal. Methods. Here, we aimed to understand the molecular basis of AMR in 66 S. Typhi organisms isolated in a cross-sectional study performed in a suburb of Chandigarh in Northern India using whole-genome sequencing and phylogenetic analysis. Results. We identified 7 S. Typhi organisms with the R717Q mutation in the acrB gene that was recently found to confer resistance to azithromycin in Bangladesh. Six out of the seven azithromycin-resistant S. Typhi isolates also exhibited triple mutations in gyrA (S83F and D87N) and parC (S80I) genes and were resistant to ciprofloxacin. These contemporary ciprofloxacin/azithromycin-resistant isolates were phylogenetically distinct from each other and from those reported from Bangladesh, Pakistan, and Nepal. Conclusions. The independent emergence of azithromycin-resistant typhoid in Northern India reflects an emerging broader problem across South Asia and illustrates the urgent need for the introduction of typhoid conjugate vaccines in the region.

For the first time in the world, we report Enteroaggregative E.coli harbouring blaNDM−4 and blaNDM−7 that were isolated from healthy children. NDM-4 and NDM-7 are associated with increased carbapenemase activity. Clonal spread, inter-strain and interspecies horizontal transfer of such resistant determinants in a healthy population will be a cause of concern.